Secretion optimized microorganism

Abstract

Proteins having a cofactor can be secreted in an improved manner in a microorganism belonging to the genus Corynebacterium provided that the microorganism contains a nucleic acid sequence which is not naturally present in it and which comprises at least the following sequence sections: a) nucleic acid sequence coding for a protein which contains a cofactor, and b) a nucleic acid sequence which is at least 20% identical to the sequence given in SEQ ID NO. 1 or a nucleic acid sequence which is a structural homologue to this sequence, wherein the amino acid sequence which is encoded by the nucleic acid sequence b) functionally interacts with the amino acid sequence encoded by the nucleic acid sequence a) in such a manner that at least the amino acid sequence encoded by the nucleic acid sequence a) is excreted by the microorganism.

1 . Microorganism comprising a nucleic acid sequence not naturally present in it, wherein the sequence comprises:
 a) nucleic acid sequence coding for a protein that comprises a cofactor, and   b) nucleic acid sequence that is at least 20% identical to the sequence stated in SEQ ID NO. 1 or is a structurally homologous nucleic acid sequence to this sequence,   wherein the amino acid sequence coded by the nucleic acid sequence b) functionally interacts with the amino acid sequence coded by the nucleic acid sequence a) in such a way that at least the amino acid sequence coded by the nucleic acid sequence a) is secreted from the microorganism, with the proviso that the microorganism belongs to the genus  Corynebacterium.      
     
     
         2 . Microorganism according to  claim 1  wherein the folding of the amino acid sequence coded by the nucleic acid sequence a) occurs in the cytoplasm of the microorganism. 
     
     
         3 . Microorganism according to  claim 1  wherein it secretes at least the amino acid sequence coded by nucleic acid sequence a) together with at least one cofactor. 
     
     
         4 . Microorganism according to  claim 1  wherein the cofactor of the protein which nucleic acid sequence a) codes is a coenzyme or a prosthetic group. 
     
     
         5 . Microorganism according to  claim 1  wherein the amino acid sequence coded by nucleic acid sequence b) is a signal sequence for Tat secretion path. 
     
     
         6 . Microorganism according to  claim 1  wherein the amino acid sequence coded by nucleic acid sequence b) and the amino acid sequence coded by nucleic acid sequence a) are components of the same polypeptide chain. 
     
     
         7 . Microorganism according to  claim 1  wherein it is chosen from  Corynebacterium ammoniagenes  ( Brevibacterium ammoniagenes ),  Corynebacterium glutamicum, Brevibacterium taipei, Micrococcus glutamicus, Brevibacterium roseum, Brevibacterium flavum, Corynebacterium herculis, Brevibacterium lactofermentum, Corynebacterium acetoacidophilum, Brevibacterium divaricatum, Brevibacterium saccharolyticum, Brevibacterium immariophilium, Microbacterium ammoniaphilum, Corynebacterium lilium, Corynebacterium callunae, Brevibacterium thiogenitalis, Corynebacterium afermentans, Corynebacterium amycolatum, Corynebacterium auris, Corynebacterium atypicum, Corynebacterium bovis, Corynebacterium callunae, Corynebacterium casei, Corynebacterium confusum, Corynebacterium diphtheriae, Corynebacterium equi, Corynebacterium halotolerans, Corynebacterium hanseni, Corynebacterium glucuronolyticum, Corynebacterium jeikeium, Corynebacterium minutissimum, Corynebacterium mycetoides, Corynebacterium nigricans, Corynebacterium pseudodiptheriticum, Corynebacterium pseudotuberculosis, Corynebacterium resistens, Corynebacterium striatum, Corynebacterium tuscaniae, Corynebacterium tuscaniense, Corynebacterium ulcerans, Corynebacterium urealyticum , or  Corynebacterium xerosis.    
     
     
         8 . Process for preparation of a protein having a cofactor by a microorganism belonging to the genus  Corynebacterium , the process comprising the process steps:
 a) inserting a nucleic acid sequence not naturally present in the microorganism, the nucleic acid sequence comprising the following sequence segments—
 i) nucleic acid sequence coding for a protein that comprises a cofactor, and 
 ii) nucleic acid sequence that is at least 20% identical to the sequence stated in SEQ ID NO. 1 or is a structurally homologous nucleic acid sequence to this sequence, 
 into a microorganism, wherein sequence segments i) and ii) are functionally coupled, 
   b) expressing the nucleic acid sequence according to a) in the microorganism.   
     
     
         9 . Process according to  claim 8  wherein the amino acid sequence coded by nucleic acid sequence a) is secreted from the microorganism together with at least one cofactor. 
     
     
         10 . Process according to  claim 8  wherein the cofactor of the protein which the nucleic acid sequence a) codes is a coenzyme or a prosthetic group. 
     
     
         11 . Process for preparation of a protein comprising a cofactor comprising the process step of cultivating a microorganism according to  claim 1 , wherein the microorganism secretes the protein into the medium surrounding the microorganism. 
     
     
         12 . Process according to  claim 8  wherein the protein is an enzyme chosen from redox-enzyme, oxidase, peroxidase, hydrogenase, dehydrogenase, reductase, biotin-dependent enzyme, CO 2 -fixing enzyme, protease, amylase, cellulase, lipase, hemicellulase, pectinase, mannanase or combinations thereof.